Inappropriate establishment of fetal and maternal vascular beds within the placenta results in compromised fetal health; however, an understanding of the molecular determinants that underlie the development of placental vasculature are far from complete. Genetic deletion of Distal-less (Dlx) 3 in mouse models is embryonic lethal at E9.5-E10 due to putative failure of the placental labyrinth to expand and undergo proper vascular morphogenesis. The central hypothesis addressed in this R21 application is that Dlx3 plays a critical role in the developmental determination of the trophoblast population within the placental labyrinth that facilitates formation and expansion of the fetal vascular compartment. To examine our central hypothesis, it is necessary to adopt new methodologies for imaging and quantifying placental vascular organization and blood flow. Here, we propose to use in vivo and ex vivo two-photon excited fluorescence (2PEF) microscopy to study the topology and organization of placental vascular beds and the functional consequences of any vascular differences on fetal blood flow for different Dlx3 genotypes. Specific Aims are: Aim 1. To optimize the use of two-photon excited fluorescence microscopy to image vascular compartments in the developing mouse placenta and examine the hypothesis that Dlx3 is an important determinant of angiogenic potential of the mouse placental labyrinth. Aim 1 will optimize experimental protocols for the ex vivo examination of fetal vascular bed topology and collagen matrix organization in mice using 2PEF microscopy. By iteratively imaging and removing tissue through optical ablation, we will visualize the vasculature of the entire placental disk at different gestational ages. Dlx3+/+,Dlx3+/- and Dlx3-/-mice expressing the Tie2-GFP transgene specifically in the fetal vascular compartment together with fluorescent labeling of the maternal vessels/blood spaces will allow simultaneous imaging and quantification of both maternal and fetal vascular compartments. These imaging studies provide the unique opportunity to evaluate the effect(s) of loss of Dlx3 on placental labyrinth vessel number and volume/surface area as well as vessel connectivity, tortuiosity and branching morphogenesis. Aim 2. To examine the hypothesis that in vivo blood flow is reduced in the Dlx3-/- implantation sites. In Aim 2, we shift to in vivo examination of the kinetics of blood flow within the labyrinth in the presence and absence of Dlx3. 2PEF imaging of individual blood vessels enables blood flow speed to be studied one vessel at a time within the labyrinth. We will determine the effects of genetic perturbation of Dlx3 on in vivo blood flow in the fetal vascular compartment and connect these changes to differences in vascular topology determined in Aim 1. The long term goal of the proposed studies is to develop exciting new imaging strategies to examine placental defects in mice representing models of human disease. Inappropriate establishment of fetal and maternal vascular beds within the placenta results in compromised health. Conditions can range from immediate, often life threatening health issues for the mother and baby, such as with preeclampsia, to the longer term effects of the uterine environment on adult onset of disease. These disease situations can have devastating effects on the patient and baby; however, an understanding of the molecular determinants that underlie the development of placental vasculature are far from complete. The studies proposed in this application optimize and implement a novel and exciting in vivo imaging strategy (two-photon excited fluorescence microscopy) to characterize and quantitate vascular bed topology, vascular fibrillar collagen matrix organization and blood flow in an important mouse model of placental failure. [unreadable] [unreadable] [unreadable]